Dystrophinopathies (Duchenne and Becker Muscular Dystrophies)

Background Histopathology & Immunohistochemistry Differential Diagnosis

BACKGROUND AND CLINICAL INFORMATION: Head

Summary Dystrophin-Glycoprotein Complex (DGC) Biochemical Diagnosis Clinical Features

Useful Web Sites: Muscular Dystrophy Association (MDA) web site

Summary: Dystrophinopathies are X-linked recessive muscular dystrophies associated with abnormal dystrophin coded by the dystrophin gene on chromosome Xp21.2. Duchenne muscular dystrophy should be regarded as the more severe form and Becker muscular dystrophy should be regarded as a mild allelic variant of Duchenne muscular dystrophy. Multiplex PCR analysis is useful for molecular diagnosis. The histologic picture is that of muscular dystrophy and typically with small clusters of necrotic fibers found at the periphery of the muscle fascicle. The lack of dystrophin can be evaluated by western blot and immunostaining.

Incidence: Duchenne muscular dystrophy occurs at a frequency of 1/3500 life born male.

Genetics: The dystrophin gene (2.3 Mb, about 1% of the entire X chromosome) on chromosome Xp21.2 is one of the largest genes that has been identified thus far. The coding sequences comprise only 0.6% of the gene.

Molecular diagnosis: Multiplex PCR analysis of 18 exons in total will detect 98% of all the Duchenne and Becker muscular dystrophy. Southern blots were used in the past for diagnosis.

Carrier states: Carriers can be detected by serum CK level, molecular, biochemical and immunohistochemical methods.

Serum creatine kinase (CK) level is very high particularly during the early clinical course, may be in the range of 200-300 times that of normal. Elevated CK is very useful for the screening of preclinical cases.

Dystrophin-glycoprotein complex (DGC): The DGC is an oligomeric complex composed of dystrophin and three major subcomplexes that are composed of dystrophin associated proteins:

Dystrophin:

· Dystrophin is on the cytoplasmic aspect and has four functional domains, the N-terminal, the C-terminal, the cysteine-rich-domain and the rod-domain.

· The N-terminal binds with actin-cytoskeleton; C-terminal binds with syntrophin complex; Cystein-rich region (near C-terminal) probably binds with dystroglycan complex.

· Most abundant at the neuromuscular junction.

· Dystroglycan complex: a- and b-dystroglycan are derived from the same precursor molecular and they links dystrophin with laminin. Deficiency of dystroglycan has almost never been linked with human disease.

a-dystroglycan links the membrane to sarcolema to the basement membrane

b-dystroglycan binds with dystrophin.

Sarcoglycan complex: a-, b-, g- and d-sarcoglycans; a membrane complex and its functions are not clear.

Syntrophin complex: a-, b1-, and b2-syntrophins and dystrobrevin; binds to the C-terminal of dystrophin. Deficiency of syntrophins has not been shown to be linked with human disease.

Biochemical diagnosis:

· The amount of dystrophin can be assessed by western blots and the amount of residual dystrophin correlates well with the disease severity.

· In Duchenne muscular dystrophy patients, antibodies directed against C-terminal detect absence of dystrophin and antibodies directed against eh N-terminal or rod domain detect truncated dystrophin species in a decreased amount. Antibodies against C-terminal are particularly useful for the separation of Duchenne and Becker muscular dystrophy.

· In Becker muscular dystrophy, antibodies against either N- or C-terminal detect a reduced amount of dystrophin of abnormal size.

Clinical features: Duchenne Becker

Duchenne:

· Age of onset: First symptoms are mostly seen before 5 years of age, most commonly before 3 years of age. Lost of ambulation usually occurs between 7 to 11 years of age.

· Course: Most patient die before the age of 20 in the past but patients are having better survivals recently because of improved care. Despite the best effort, however, most patient will died in or before the 4^th decade.

· Muscular Symptoms: Typically no abnormalities are awared by the parents until the child starts walking. Muscle weakness is the cardinal problem. Most common initial symptoms are abnormal gait, frequent falls and difficulty in climbing steps. A typical waddling gait on a wide base is usually combined with a tendency to walk on the toes and associated with a lumbar lordosis. The child will invariably need at least two seconds to get up from a sitting position on the floor (normal child is one second) and Gower’s manoeuvre (due to muscle weakness of pelvic girdle and proximal leg muscle) is seen in the later stage. Pseudohypertrophy of calf muscle is seen in the more advanced cases.

· Deformities: Fixed deformities are uncommon as far as the child remains ambulant. However, rapid development of fixed deformities related to their habitual posture occurs once the child loss the ability to walk.

· Intelligence: Duchenne patients have a mean IQ of 85 but many of them also have normal intelligence. About one-thrid of the patients have subnormal intelligence which is non-progressive.

· Respiratory: Respiratory deficit at early stage of disease and respiratory failure at the later stage of disease.

· Cardiac involvement: Cardiac involvement is usually asymptomatic but can be documented by EKG. There is a steady decline in left ventricular function that may be correlated with the rate of decline in skeletal muscle function in some but not all cases.

· Smooth muscle involvement: Minimal, if any.

· EMG: Myopathic pattern.

Becker:

· Summary: Becker muscular dystrophy is identical to Duchenne muscular dystrophy in many aspects except that it is clinically less severe and has later onset (mean age of onset is 11 years).

· Salient distinction: Patients with Becker muscular dystrophy are able to walk after 16 years of age but patients with Duchenne muscular dystrophy will not be able to walk after 13 years of age. Albeit, some patients will eventually become wheelchair bound.

HISTOPATHOLOGY AND IMMUNOHISTOCHEMISTRY: Head

Muscle Histology Immunohistochemistry Cardiac Pathology

Muscle Histology:

· Stages: Myopathic changes in early stages, fibrosis and near total loss of muscle in end stages.

· Excessive variation in fiber size may be the earliest histologic findings in pre-clinical cases.

· Necrotic and fibers in small groups: Segmental necrosis of muscle cells with an increased and not uncommon dramatic amount of contraction artifact is very common. Small clusters of necrotic fibers (3-10 fibers) are often characteristically found at the periphery of the fascicle. The earlier stages of the necrotic fibers are pale and enlarged.

· Fiber hypertrophy: Abnormally large rounded and hypertrophic fibers are common.

· Hypercontraction artifact: The amount of hypercontraction artifact in formalin fixed paraffin sections is usually dramatic. Delta lesions are common.

· Regenerating fibers, often smaller than normal, are present and are more common in early stages. They also tend to occur in small groups.

· Randomly distributed small fibers probably resulted from incomplete regeneration rather than shrinkage are present.

· Calcium depositions are often associated with necrotic fibers.

· Hyaline fibers: Unusually large, eosinophilic and perfectly round fiber with a ground glass cytoplasm, resulted from segmental hypercontraction of myofibers. These fibers stain darkly with all stain.

· Macrophages may be present.

· Distinctive pattern of collagen accumulation: Interstitial fibrosis is usually pattern. Collagen often accumulates as small bundles that are parallel to each other and also parellel to the muscle fibers. These collagen bundles are best seen with semithin sections or electron microscopy.

· Internally situated nuclei are not common.

· Muscle spindle: No necrosis.

· Extraocular muscle: No necrosis.

Muscle Immunohistochemistry:

· Antibodies for C-terminal, N-terminal, and mid-rod should be used (Novacastra).

· Normal muscle: dystrophin appears as a thin continuous sarcolemmal staining that is present in every individual fiber.

· Absence of dystrophin on sarcolema is a feature of Duchenne muscular dystrophy. Sporadiac fibers that express dystrophin, so-called revertant fibers, are fibers that may possibly have a “corrective” mutation that makes the dystrophin gene competent. Mosaicism is seen in carriers.

· Marked reduction in a- and b-dystroglycans and all sarcoglycans is seen in Duchenne muscular dystrophy; a phenomenon that is secondary to distrophin deficiency.

· Expression of dystrophin in Becker muscular dystrophy is patchy.

· Up regulation of utrophin. Utrophin (utrophin gene is on chromosome 6) is a close analogue of dystrophin and is expressed normally only at the postjunctional sarcolemma at the crest of the junctional folds at the neuromuscular junction and is attached to dystrophin-associated proteins.

Cardiac Pathology: Fibrosis in the left ventricle.

DIFFERENTIAL DIAGNOSIS: Head

Dystrophinopathies vs. limb girdle dystrophy: Particlulary LGMD 2C and 2D (formerly known as severe childhood autosomal recessive muscular dystrophy or SCARMD). Duchenne and Becker muscular dystrophy also present with limb girdle like weakness. The genetics, clinical symptoms, and molecular diagnosis are different.